Pharmacological modulation of the cAMP signaling of two isoforms of melanocortin-3 receptor by melanocortin receptor accessory proteins in the tetrapod Xenopus laevis

As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals and its pharmacological effect could be directly influenced by the presence of melanocortin accessory proteins, MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not been thoroughly evaluated yet. In this work, we performed sequence alignment, phylogenetic and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.

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Pharmacological modulation of dual melanocortin‐4 receptor signaling by melanocortin receptor accessory proteins in the Xenopus laevis

Journal of Cellular Physiology ◽

10.1002/jcp.30280 ◽

2021 ◽

Author(s):

Lei Li ◽

Ying Xu ◽

Jihong Zheng ◽

Zhe Kuang ◽

Cong Zhang ◽

...

Keyword(s):

Xenopus Laevis ◽

Melanocortin Receptor ◽

Accessory Proteins ◽

Receptor Signaling ◽

Pharmacological Modulation ◽

Melanocortin 4 Receptor

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Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by C Terminus of MRAP2 Protein

10.21203/rs.3.rs-829492/v1 ◽

2021 ◽

Author(s):

Meng Wang ◽

Yue Zhai ◽

Xiaowei Lei ◽

Jing Xu ◽

Bopei Jiang ◽

...

Keyword(s):

Energy Homeostasis ◽

Transmembrane Protein ◽

Melanocortin Receptor ◽

Pharmacological Modulation ◽

C Terminus ◽

Melanin Concentrating Hormone ◽

The Central Nervous System

Abstract Background: Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts at its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate energy homeostasis and body weight. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine pivot of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal appetite and peripheral energy homeostasis. However, the regulatory and relationship between MCHR1 and MRAP2 is unknown.Results: In this study, we show that MRAP2 interacts with MCHR1 and suppresses MCHR1 signaling in vitro. We also identified the C-terminal domains of MRAP2 protein required for pharmacological modulation of intracellular Ca2+ cascades and membrane transport.Conclusions: These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

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Melanocortin receptor accessory proteins in adrenal gland physiology and beyond

Journal of Endocrinology ◽

10.1530/joe-12-0501 ◽

2013 ◽

Vol 217 (1) ◽

pp. R1-R11 ◽

Cited By ~ 23

Author(s):

T V Novoselova ◽

D Jackson ◽

D C Campbell ◽

A J L Clark ◽

L F Chan

Keyword(s):

Adrenal Gland ◽

Energy Homeostasis ◽

Functional Expression ◽

Surface Expression ◽

Vital Role ◽

Melanocortin Receptor ◽

Exocrine Function ◽

Zona Fasciculata ◽

Accessory Factor

The melanocortin receptor (MCR) family consists of five G-protein-coupled receptors (MC1R–MC5R) with diverse physiological roles. MC1R controls pigmentation, MC2R is a critical component of the hypothalamic–pituitary–adrenal axis, MC3R and MC4R have a vital role in energy homeostasis and MC5R is involved in exocrine function. The melanocortin receptor accessory protein (MRAP) and its paralogue MRAP2 are small single-pass transmembrane proteins that have been shown to regulate MCR expression and function. In the adrenal gland, MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency, where inactivating MRAP mutations account for ∼20% of cases. MRAP is highly expressed in both the zona fasciculata and the undifferentiated zone. Expression in the undifferentiated zone suggests that MRAP could also be important in adrenal cell differentiation and/or maintenance. In contrast, the role of adrenal MRAP2, which is highly expressed in the foetal gland, is unclear. The expression of MRAPs outside the adrenal gland is suggestive of a wider physiological purpose, beyond MC2R-mediated adrenal steroidogenesis.In vitro, MRAPs have been shown to reduce surface expression and signalling of all the other MCRs (MC1,3,4,5R). MRAP2 is predominantly expressed in the hypothalamus, a site that also expresses a high level of MC3R and MC4R. This raises the intriguing possibility of a CNS role for the MRAPs.

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Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by C Terminus of MRAP2 Protein

10.21203/rs.3.rs-829492/v2 ◽

2021 ◽

Author(s):

Meng Wang ◽

Yue Zhai ◽

Xiaowei Lei ◽

Jing Xu ◽

Bopei Jiang ◽

...

Keyword(s):

Energy Homeostasis ◽

Transmembrane Protein ◽

Melanocortin Receptor ◽

Pharmacological Modulation ◽

C Terminus ◽

Melanin Concentrating Hormone ◽

The Central Nervous System

Abstract Background: Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts at its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate energy homeostasis and body weight. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine pivot of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal appetite and peripheral energy homeostasis. However, the regulatory and relationship between MCHR1 and MRAP2 is unknown.Results: In this study, we show that MRAP2 interacts with MCHR1 and suppresses MCHR1 signaling in vitro. We also identified the C-terminal domains of MRAP2 protein required for pharmacological modulation of intracellular Ca2+ cascades and membrane transport. Conclusions: These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

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Pharmacological modulation of MRAP2 protein on melanocortin receptors in the sea lamprey

Endocrine Connections ◽

10.1530/ec-19-0019 ◽

2019 ◽

Vol 8 (4) ◽

pp. 378-388 ◽

Cited By ~ 7

Author(s):

Ming Zhu ◽

Bingxin Xu ◽

Meng Wang ◽

Shangyun Liu ◽

Yue Zhang ◽

...

Keyword(s):

Surface Expression ◽

Sea Lamprey ◽

Melanocortin Receptor ◽

Regulatory Function ◽

Carboxyl Terminus ◽

Melanocortin Receptors ◽

Melanocortin System ◽

Pharmacological Modulation ◽

Primitive Species ◽

First Time

Melanocortin receptors (MCRs) and their accessory proteins (MRAPs) evolutionarily first appear in the genome of sea lamprey. The most ancient melanocortin system consists of only two melanocortin receptors (slMCa and slMCb) and one MRAP2 (slMRAP2) protein, but the physiological roles have not been fully explored in this primitive species. Here, we synthesize and characterize the pharmacological features of slMRAP2 protein on two slMCRs. Our results show that the slMRAP2 protein lacks the long carboxyl terminus; it directly interacts and decreases the surface expression but enhances the α-MSH-induced agonism of slMCa and slMCb. In comparison with higher organisms such as elephant shark and zebrafish, we also demonstrate the constantly evolving regulatory function of the carboxyl terminus of MRAP2 protein, the unique antiparallel topology of slMRAP2 dimer and the homo- and hetero-dimerization of two slMCRs. This study elucidates the presence and modulation of melanocortin receptor by the accessory protein of the agnathans for the first time, which provides a better insight of the melanocortin system in ancient species of chordates.

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Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signalling and obesity

Journal of Endocrinology ◽

10.1677/joe.0.1720411 ◽

2002 ◽

Vol 172 (3) ◽

pp. 411-421 ◽

Cited By ~ 196

Author(s):

LE Pritchard ◽

AV Turnbull ◽

A White

Keyword(s):

Energy Homeostasis ◽

Proteolytic Enzymes ◽

Energy Requirement ◽

Proteolytic Processing ◽

Melanocortin Receptor ◽

Current Evidence ◽

Appetite Control ◽

Pomc Gene

Bioactive peptides derived from the prohormone, pro-opiomelanocortin (POMC), are generated in neurons of the hypothalamus and act as endogenous ligands for the melanocortin-4 receptor (MC4R), a key molecule underlying appetite control and energy homeostasis. It is therefore important to understand many aspects of POMC gene regulation in the brain, as pharmacological manipulation of POMC expression/processing could be a potential strategy to combat obesity. Most studies that have analysed POMC gene expression in the hypothalamus have focused on gene transcription experiments. Ultimately, however, factors that regulate post-translational processing and secretion of peptides will have most bearing on melanocortin signalling. This article focuses on (a) current evidence that POMC is involved in obesity, (b) how POMC transcription is regulated in the hypothalamus, (c) the mechanism by which proteolytic processing of POMC is controlled in the hypothalamus and what peptides are produced and (d) which POMC-derived peptides are the most potent ligands at the melanocortin receptor in vitro and in vivo. It seems that post-translational cleavage of POMC in the hypothalamus may be regulated with respect to energy requirement. We predict that further research into hypothalamic POMC processing, and the proteolytic enzymes involved, may yield important new clues on how flux through the MC4R pathway is regulated.

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Depletion of Alpha-Melanocyte-Stimulating Hormone Induces Insatiable Appetite and Gains in Energy Reserves and Body Weight in Zebrafish

Biomedicines ◽

10.3390/biomedicines9080941 ◽

2021 ◽

Vol 9 (8) ◽

pp. 941

Author(s):

Yang-Wen Hsieh ◽

Yi-Wen Tsai ◽

Hsin-Hung Lai ◽

Chi-Yu Lai ◽

Chiu-Ya Lin ◽

...

Keyword(s):

Body Weight ◽

Energy Homeostasis ◽

Body Weight Gain ◽

Genetically Engineered ◽

Melanocortin Receptor ◽

Synthetic Analog ◽

Melanocortin System ◽

Releasing Hormone ◽

Melanocyte Stimulating Hormone ◽

Stimulating Hormone

The functions of anorexigenic neurons secreting proopiomelanocortin (POMC)/alpha-melanocyte-stimulating hormone (α-MSH) of the melanocortin system in the hypothalamus in vertebrates are energy homeostasis, food intake, and body weight regulation. However, the mechanisms remain elusive. This article reports on zebrafish that have been genetically engineered to produce α-MSH mutants, α-MSH−7aa and α-MSH−8aa, selectively lacking 7 and 8 amino acids within the α-MSH region, but retaining most of the other normal melanocortin-signaling (Pomc-derived) peptides. The α-MSH mutants exhibited hyperphagic phenotypes leading to body weight gain, as observed in human patients and mammalian models. The actions of several genes regulating appetite in zebrafish are similar to those in mammals when analyzed using gene expression analysis. These include four selected orexigenic genes: Promelanin-concentrating hormone (pmch), agouti-related protein 2 (agrp2), neuropeptide Y (npy), and hypothalamic hypocretin/orexin (hcrt). We also study five selected anorexigenic genes: Brain-derived neurotrophic factor (bdnf), single-minded homolog 1-a (sim1a), corticotropin-releasing hormone b (crhb), thyrotropin-releasing hormone (trh), and prohormone convertase 2 (pcsk2). The orexigenic actions of α-MSH mutants are rescued completely after hindbrain ventricle injection with a synthetic analog of α-MSH and a melanocortin receptor agonist, Melanotan II. We evaluate the adverse effects of MSH depletion on energy balance using the Alamar Blue metabolic rate assay. Our results show that α-MSH is a key regulator of POMC signaling in appetite regulation and energy expenditure, suggesting that it might be a potential therapeutic target for treating human obesity.

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An interaction between dorsal and ventral regions of the marginal zone in early amphibian embryos

Development ◽

10.1242/dev.56.1.283 ◽

1980 ◽

Vol 56 (1) ◽

pp. 283-299

Author(s):

J. M. W. Slack ◽

D. Forman

Keyword(s):

Xenopus Laevis ◽

Salt Solution ◽

Direct Contact ◽

Marginal Zone ◽

Normal Development ◽

Large Area ◽

Amphibian Embryos ◽

In Vitro Interaction

When small explants from early gastrulae of Xenopus laevis are allowed to develop in abuffered salt solution there is a considerable difference between the patterns of differentiation obtained from different dorsoventral levels of the marginal zone. These patternsof differentiation correspond to the fates of the different regions in the course of normal development. They are not altered if several explants of the same type are fused before culture. If a ventral marginal zone explant from Xenopus is cultured in contact with a piece of dorsal marginal zone from the axolotl, it forms structures more dorsal in character than it would in isolation or in normal development. This behaviour is shown only feebly with other regions of the axolotl gastrula. A piece of dorsal marginal zone from Xenopus is not affected in its development by culture in contact with an explant of ventral marginal zone from the axolotl. The dorsalization of ventral marginal zone explants is shown only if there is a large area of direct contact with the dorsal explant and if the pieces remain in contact for a period of 48 h or more. It is proposed that this in vitro interaction is the same as the dorsoventral component of action of the ‘organizer’ graft discovered by Spemann and Mangold.

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OR04-01 MRAP2 Regulates Energy Homeostasis by Promoting Primary Cilia Localization of MC4R

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1166 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Adelaide A Bernard ◽

Irene Ojeda Naharros ◽

Florence Bourgain Guglielmetti ◽

Xinyu Yue ◽

Christian Vaisse

Keyword(s):

Body Weight ◽

Primary Cilium ◽

Energy Homeostasis ◽

Primary Cilia ◽

Small Population ◽

The Body ◽

Melanocortin Receptor ◽

Hypothalamic Neurons

Abstract Genetic studies in humans and mice have demonstrated that the Melanocortin 4 Receptor (MC4R) is essential for adequate regulation of food intake and body weight. MC4R is expressed in a small population of hypothalamic neurons and very little is known about its molecular and cellular dynamics in vivo. We have recently demonstrated that MC4R localizes to and functions at the primary cilia of select hypothalamic neurons to control energy homeostasis. The primary cilium is a solitary hair-like organelle that serves as an antenna sensing extracellular environment. Defective primary cilia lead to a series of conditions known as ciliopathies, that can manifest through a variety of clinical features, including hyperphagia and obesity. Here we establish that the ciliary localization and the body weight regulating activity of MC4R is dependent on a single-pass transmembrane accessory protein: the Melanocortin Receptor Associated Protein 2 (MRAP2). Specifically, we show that deleting MRAP2 specifically from MC4R neurons (MC4RMRAP2-/-) leads to early onset obesity and hyperphagia. In vitro, co-expression of MRAP2 in ciliated IMCD3 cells increases MC4R localization to the primary cilium. We further demonstrate that MRAP2 and MC4R colocalize specifically at the primary cilium in vivo, and that MC4R fails to localize to the primary cilium when MRAP2 is deleted. These findings highlight the role of the primary cilium in the control of energy homeostasis, and the importance of accessory proteins for the localization of GPCRs to the primary cilium where they exert their function, in this case being critical for the regulation of energy homeostasis.

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